RNA-seq as a diagnostic tool

Because of the development of low-cost, high-throughput next-generation sequencing technology, RNA sequencing (RNA-seq) has arisen as a novel technique in genetic diagnostic laboratories. RNA-seq enables the qualitative and quantitative identification of genome-wide changes in RNA expression in clinical samples, and it is increasingly being employed as a diagnostic complement for whole-exome and whole-genome sequencing.

Professor Brendan Lee of Baylor College of Medicine in Houston, Texas, USA, published a review in JAMA titled "RNA Sequencing as a Diagnostic Tool", briefly describing how RNA-seq works, important considerations, the value of RNA-seq, and the evidence base to improve clinical practitioners' understanding of RNA-seq in the field of clinical diagnosis.

The study begins by explaining how RNA-seq works.

1. Select and collect tissue samples
2. Isolate and purify mRNA or total RNA
3. Convert RNA to cDNA by reverse transcription
4. Add sequencing adaptors and establish sequencing library
5. Perform high-throughput parallel sequencing and mapping to reference genome

The selection of the right tissue type is a crucial aspect for RNA-seq analysis. Because gene expression and splicing patterns differ by tissue and cell type, and disease-associated tissues are the ideal samples for RNA-seq. Clinically accessible tissue samples, such as blood, skeletal muscle, and skin, may differ in gene expression significantly from true focal tissue samples, resulting in incorrect sequencing analysis results.

Another critical factor to consider when performing RNA-seq analysis is whether aberrant splicing impacts protein function. Normal transcription products may be lost as a result of abnormal splicing, and in certain situations, numerous splicing products may be produced. In the same way, aberrant splicing patterns inside the frame may have no influence on protein function. As a result, protein function tests may still be required to explain the toxicity of particular splice variants.

There are currently no evidence-based clinical guidelines for using RNA-seq to diagnose uncommon diseases or malignancies. Clinical diagnostic laboratories that offer RNA testing typically employ it to identify the functional impact of DNA variants of uncertain significance, which are most commonly used in cancer genetics. Additional illness or benign mutations may alter clinical application guidelines. RNA-seq has been used effectively in the scientific community to detect rare monogenic genetic disorders.

In conclusion, RNA sequencing can increase molecular diagnosis rates by diagnostic exome or whole genome sequencing. Due to insufficient annotation of some DNA variants, RNA-seq can successfully discover pathogenic mutations. The application of RNA-seq in clinical diagnosis necessitates the development and standardization of analytical methodologies.

Across the industry there are CROs offering RNA-seq solutions to expedite the process and enhance the accuracy, such as Creative Biolabs, which can accommodate whole genome sequencing (WGS), whole exome sequencing (WES), targeted sequencing, whole transcriptome sequencing (WTS), immune repertoire squencing (Rep-Seq), epigenetic modifications, 3D configuration of the genome, metagenomics sequencing, ctDNA sequencing, single cell sequencing, and spatial transcriptome sequencing, taking the advantageous SuPrecision™ platform.